The overall goal of this grant is to understand why polyamines are essential to the migration of GI mucosal cells during the early mucosal restitution component of healing. Experiments supported during the past 15 years this project has been active have shown that: 1) polyamines are essential to the healing of gastric and duodenal mucosa, 2) polyamines are required for mucosal restitution which depends on cell migration, 3) polyamines are essential to cell migration, 4) polyamines are necessary for the normal functioning of several components of the cytoskeleton involved in cell migration, 5) migration of IEC-6 cells is regulated by the Rho GTPases, and 6) polyamines influence the levels and activities of the Rho GTPases. The first set of specific aims will determine the effect of polyamines on the Rho family of GTPases during the migration of IEC-6 cells. IEC-6 cells stably transfected with constitutively active and dominant negative RhoA, Rac1 and Cdc42 will be used to determine their hierarchy of activation and where the polyamines interact. Preliminary data indicate that both RhoA and Raci are essential for cell migration but that only Rac1 activity is sufficient for migration. The second set of specific aims will determine the role of polyamines in integrin signaling by examining ccli attachment and spreading. The same clones and polyamine depletion will be used to examine the roles of Rho, Rac and Cdc42. Polyamine depletion will be used to determine the role of polyamines in the distribution and phosphorylation of proteins involved in the formation of focal adhesions and in cell spreading. In the third set of specific aims a highly specific polyamine antibody will be used to determine the cellular localization of polyamines during the processes of spreading and migration and to determine the colocalization of polyamines with cytoskeletal proteins and those regulating cytoskeletal transformations. These studies will provide the first description of the involvement of the Rho-GTPases in epithelial cell migration and spreading and how the activities of these proteins are regulated by polyamines. They also will provide the first studies of polyamine localization during dynamic cell processes and define the mechanisms by which polyamines regulate those processes. This work will increase our understanding of cell function basic to many clinical conditions in which cell migration is a major component. These include peptic ulcer disease, cancer, and inflammatory bowel disease.